ACTH secretion by a mouse pituitary tumor cell line is inhibited by physiological concentrations of glucocorticoids and the effect is reversed by growing the cells in glucocorticoid free medium for several generations or by the addition of hypothalamic extract. Four immunoreactive forms of ACTH have been observed in the tumor cell line and in primry cell cultures from normal mouse pituitary. The molecular weights of these forms as determined by SDS gel electrophoresis and gel filtration in guanidine-HCl are: 31,00 (31K ACTH), 23,000 (23K ACTH), 8,000 (8K ACTH) and 4500 (4.5K ACTH). Pulse label and pulse chase experiments with radioactive amino acids show that label first enters 31K ACTH, then 23K ACTH and finally 8K ACTH and 4.5K ACTH. Labeling experiments with radioactive mannose and glucosamine show that the 31K, 23K and 8K forms of ACTH have sugars in them. The radioactive forms of ACTH were purified in these experiments by a double antibody immunoprecipitation procedure and by SDS gel electrophoresis. Studies are now being done to determine which forms of ACTH are released into the culture medium before and after treatment of tumor cell cultures and primary cell cultures from mouse pituitary with glucocorticoids, hypothalamic extracts and other agents that regulate ACTH synthesis and secretion. Cell fractionation studies are also being done to determine where the forms of ACTH are located in the cell and where conversion of the forms takes place in the cell. Messenger RNA is being isolated from tumor cells and translated in cell free systems and in xenopus oocytes to determine what the first product of ACTH synthesis is.